Team:Duke/Results

DESIGN STATEMENT

To design and express a significantly more thermostable Griffithsin dimer and monomer for future implementation in a diagnostic assay.

MODELING of GRIFFITHSIN

Our design began with taking the originally published sequence of griffithsin and modeling thermostable changes in the sequence. Click here for more information on our modeling.

DESIGNING A HIGH-EXPRESSING CONSTRUCTS

After creating an optimal amino acids sequence from the Yasara modeling, we codon optimized the sequence in E. coli via IDT (link here). To express this protein maximum level, we placed the gene within a T7 promoter system—an adapted viral promoter that maximizes expression. This gBlock was subsequently cloned into our the LC-pSmart Kan vector, using Gibson Assembly.

IPTG INDUCED EXPRESSION

To mimic industrial expression protocols, we created a protocol to first maximize the biomass of the Griffithsin-expressing cells. Then the IPTG was added to turn on the promoter and hijack the cell’s metabolism to producing GRFT. The GRFT was then removed from the cells.

To express griffithsin (GRFT) at maximum levels, dynamic metabolic control was exercised using IPTG induction as the metabolic valve.

  1. Biomass is grown in of SM10++(max 10g/L) media for 18 hours
  2. IPTG rich SM0++ media is then added to the culture which induces:
    1. The vast majority of its metabolism shifts to protein creation as the strong promoter is turned on.
    2. Cell growth will dramatically decrease
  3. Homgenization
    1. Since Griffithsin is an intracellular protein, homogenization was chosen as the method taken to lyse the cells.
    2. Homogenization feeds a sample through small aperture where the cell is lysed by shearing

CONFIRMATION OF GRIFFITHSIN EXPRESSION

We ran an SDS page gel on the lysate to confirm expression of griffithsin. The protocol we used for these is in the experiments tag but it is also diagramed below.

The following gel shows the presence of griffithsin.

Figure 1: 12% Bis-Tris SDS-PAGE. Lanes: NEB 10-200 kDa Ladder Ladder (L), Empty pSmart Vector (EV), Thermostable Griffithsin Monomer (TSm), Thermostable Griffithsin Dimer (TS)

Using this construct, the only insert we found was the 9bp of pSB1C3 homology (Figure 3).

Figure 3: pSB1C3 self-homology is in empty vectors

Sequencing the vectors that were empty from colony PCR, we found that unanimously that in place of the gene insert was the 9bp pSB1C3 homology instead.

Hydropathicity Considerations

Previous studies in our lab produced the following SDS-PAGE gel confirming that Wildtype-Griffithsin runs at 12 kDa.